NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia.

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

Unraveling astrocyte behavior in the space brain: Radiation response of primary astrocytes.

Introduction: Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes' response toward low linear energy transfer (LET) X-rays and high-LET carbon (12C) and iron (56Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Since astronauts are exposed to ionizing radiation and microgravity during space missions, the effect of simulated microgravity on DNA damage induction and repair was investigated. Methods: Primary murine cortical astrocytes were irradiated with different doses of X-rays, 12C and 56Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes' reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X- rays were investigated by mRNA-sequencing. RT-qPCR for several genes of interest was performed with RNA from X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1ß, Il6, and Tgfß1. Levels of the pro inflammatory cytokine IL-6 were determined using ELISA. DNA damage response was investigated after exposure to X-rays followed by incubation on a 2D clinostat to simulate the conditions of microgravity. Results: Astrocytes showed distinct responses toward the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSBs) and the respective repair was dose-, LET- and time-dependent. Simulated microgravity had no significant influence on DNA DSB repair. Proliferation and cell cycle progression was not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. Discussion: In conclusion, primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types.

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies.

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson's disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y.

Rare germline variants in the E-cadherin gene CDH1 are associated with the risk of brain tumors of neuroepithelial and epithelial origin.

The genetic basis of brain tumor development is poorly understood. Here, leukocyte DNA of 21 patients from 15 families with ≥ 2 glioma cases each was analyzed by whole-genome or targeted sequencing. As a result, we identified two families with rare germline variants, p.(A592T) or p.(A817V), in the E-cadherin gene CDH1 that co-segregate with the tumor phenotype, consisting primarily of oligodendrogliomas, WHO grade II/III, IDH-mutant, 1p/19q-codeleted (ODs). Rare CDH1 variants, previously shown to predispose to gastric and breast cancer, were significantly overrepresented in these glioma families (13.3%) versus controls (1.7%). In 68 individuals from 28 gastric cancer families with pathogenic CDH1 germline variants, brain tumors, including a pituitary adenoma, were observed in three cases (4.4%), a significantly higher prevalence than in the general population (0.2%). Furthermore, rare CDH1 variants were identified in tumor DNA of 6/99 (6%) ODs. CDH1 expression was detected in undifferentiated and differentiating oligodendroglial cells isolated from rat brain. Functional studies using CRISPR/Cas9-mediated knock-in or stably transfected cell models demonstrated that the identified CDH1 germline variants affect cell membrane expression, cell migration and aggregation. E-cadherin ectodomain containing variant p.(A592T) had an increased intramolecular flexibility in a molecular dynamics simulation model. E-cadherin harboring intracellular variant p.(A817V) showed reduced ß-catenin binding resulting in increased cytosolic and nuclear ß-catenin levels reverted by treatment with the MAPK interacting serine/threonine kinase 1 inhibitor CGP 57380. Our data provide evidence for a role of deactivating CDH1 variants in the risk and tumorigenesis of neuroepithelial and epithelial brain tumors, particularly ODs, possibly via WNT/ß-catenin signaling.

A face-to-face comparison of claudin-5 transduced human brain endothelial (hCMEC/D3) cells with porcine brain endothelial cells as blood-brain barrier models for drug transport studies.

BACKGROUND: Predictive in vitro models of the human blood-brain barrier (BBB) are essential in early drug discovery and development. Among available immortalized human brain capillary endothelial cell lines (BCECs), the hCMEC/D3 cell line has become the most widely used in vitro BBB model. However, monolayers of hCMEC/D3 cells form only moderately restrictive barriers, most likely because the major tight junction protein, claudin-5, is markedly downregulated. Thus, hCMEC/D3 monolayers cannot be used for vectorial drug transport experiments, which is a major disadvantage of this model. METHODS: Here we transduced hCMEC/D3 cells with a claudin-5 plasmid and compared the characteristics of these cells with those of hCMEC/D3 wildtype cells and primary cultured porcine BCECs. RESULTS: The claudin-5 transduced hCMEC/D3 exhibited expression levels (and junctional localization) of claudin-5 similar to those of primary cultured porcine BCECs. The transduced cells exhibited increased TEER values (211 Ω cm2) and reduced paracellular mannitol permeability (8.06%/h), indicating improved BBB properties; however, the barrier properties of porcine BCECs (TEER 1650 Ω cm2; mannitol permeability 3.95%/h) were not reached. Hence, vectorial transport of a selective P-glycoprotein substrate (N-desmethyl-loperamide) was not observed in claudin-5 transduced hCMEC/D3 (or wildtype) cells, whereas such drug transport occurred in porcine BCECs. CONCLUSIONS: The claudin-5 transduced hCMEC/D3 cells provide a tool to studying the contribution of claudin-5 to barrier tightness and how this can be further enhanced by additional transfections or other manipulations of this widely used in vitro model of the BBB.

Fumaric Acids Do Not Directly Influence Gene Expression of Neuroprotective Factors in Highly Purified Rodent Astrocytes.

(1) Background: Dimethylfumarate (DMF) has been approved for the treatment of relapsing remitting multiple sclerosis. However, the mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood. Former reports suggest a neuroprotective effect of DMF mediated via astrocytes by reducing pro-inflammatory activation of these glial cells. We investigated potential direct effects of DMF and MMF on neuroprotective factors like neurotrophic factors and growth factors in astrocytes to elucidate further possible mechanisms of the mode of action of fumaric acids; (2) Methods: highly purified cultures of primary rat astrocytes were pre-treated in vitro with DMF or MMF and incubated with lipopolysaccharides (LPS) or a mixture of interferon gamma (IFN-γ) plus interleukin 1 beta (IL-1ß) in order to simulate an inflammatory environment. The gene expression of neuroprotective factors such as neurotrophic factors (nuclear factor E2-related factor 2 (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF)) and growth factors (fibroblast growth factor 2 (FGF2), platelet-derived growth factor subunit A (PDGFa), ciliary neurotrophic factor (CNTF)) as well as cytokines (tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), IL-1ß, inducible nitric oxide synthase (iNOS)) was examined by determining the transcription level with real-time quantitative polymerase chain reaction (qPCR); (3) Results: The stimulation of highly purified astrocytes with either LPS or cytokines changed the expression profile of growth factors and pro- inflammatory factors. However, the expression was not altered by either DMF nor MMF in unstimulated or stimulated astrocytes; (4) Conclusions: There was no direct influence of fumaric acids on neuroprotective factors in highly purified primary rat astrocytes. This suggests that the proposed potential neuroprotective effect of fumaric acid is not mediated by direct stimulation of neurotrophic factors in astrocytes but is rather mediated by other pathways or indirect mechanisms via other glial cells like microglia as previously demonstrated.

Immunity in Gilles de la Tourette-Syndrome: Results From a Cerebrospinal Fluid Study.

Background: Several lines of evidence support the hypothesis of an autoimmune origin of Gilles de la Tourette-Syndrome (GTS). Accordingly, in a recent study we detected positive oligoclonal bands (OCB) in cerebrospinal fluid (CSF) in >30% of adult patients indicating an intrathecal antibody synthesis. However, until today no corresponding antibodies could be identified. The aims of this study were to replicate our findings of positive OCB in an independent sample and to detect CSF autoantibodies. Methods: In this prospective study, 20 adult patients with GTS (male: female = 18:2, median age 36.1 years ± 14.34 SD) were included. All patients were thoroughly clinically characterized. Magnetic Resonance Imaging (MRI) and CSF standard measurements were performed. Isoelectric focusing on polyacrylamide gels with silver staining was used to detect OCB. To examine specific and unspecified autoantibodies, we used transfected Human Embryonic Kidney (HEK) cells expressing different surface antigens (NMDA-, CASPR2-, LGI1-, AMPA-, or GABAB1/B), indirect immunofluorescence on different brain tissue sections, and enzyme-linked visualization. Additionally, we differentiated Glioma stem cells SY5Y (human neuroblastoma) using retinoic acid and astrocytes (rat). Results: CSF analyses showed positive OCB (type 2) in 4/20 patients (20%). Using transfected HEK cells we did not find specific surface-autoantibodies. Immunohistochemistry on tissue-sections, SY5Y Glioma stem-cells, and astrocytes showed no specific binding patterns either. Conclusions: Our results corroborate previous findings and demonstrate positive OCB in a substantial number of patients with GTS (prevalence in healthy controls: 5%). Although this is the largest study investigating CSF autoantibodies in GTS using several techniques, we failed to detect any specific or unspecified autoantibodies.

Correction to: Investigation of Neuregulin-1 and Glial Cell-Derived Neurotrophic Factor in Rodent Astrocytes and Microglia.

The original version of this article unfortunately contained mistakes in the author group and affiliation sections. Author Markus H. Schwab's name was incorrectly presented as "H. Markus Schwab" and his affiliations were incorrectly assigned as "1 and 3" instead of "2 and 3".

Polarized microglia do not influence oligodendrocyte lineage cells via astrocytes.

Microglia can adopt different activation patterns, ranging from a pro-inflammatory M1- to an anti-inflammatory M2-like phenotype in which they play crucial roles in various neuroinflammatory diseases. M2-like microglia are described to drive remyelination, whereas detrimental effects have been attributed to M1-like microglia. How polarized microglia might act on oligodendrocyte lineage cells indirectly by influencing astrocytes has not been studied in detail. In this study, conditioned media from polarized murine microglia were used to treat astrocytes and astrocytic gene expression was analyzed by microarray for genes known to influence oligodendrocyte lineage cells. Supernatants of astrocytes previously stimulated with soluble effectors from polarized microglia were used to investigate effects on oligodendrocyte precursor cells (OPC). Growth factors known to induce OPC proliferation, differentiation, and survival were upregulated in astrocytes treated with supernatants from M1-like microglia while M0- and M2-like microglia only had negligible effects on the expression of these factors in astrocytes. Despite the upregulation of these factors in M1 stimulated astrocytes there were no significant effects on OPC in vitro. All astrocyte supernatants induced proliferation of A2B5+ OPC and inhibited differentiation of OPC into mature oligodendrocytes. A trend toward enhanced migration of OPC was induced by M1 stimulated astrocytes. Our data suggest that M1-like microglia may potentially influence OPC and remyelination indirectly via astrocytes by inducing the expression of respective growth factors, however, this has no significant effect in addition to the already strong effects of unstimulated astrocytes on OPC. Nevertheless, the observed effect may be of relevance in other pathophysiological scenarios.

Investigation of Neuregulin-1 and Glial Cell-Derived Neurotrophic Factor in Rodent Astrocytes and Microglia.

Growth factors play a crucial role during de- and remyelination of the central nervous system (CNS) due to their neurotrophic functions. We have previously shown that the growth factors neuregulin-1 (Nrg-1) and glial cell-derived neurotrophic factor (Gdnf) are upregulated during the first 2 weeks after induction of toxic demyelination in the CNS. Nevertheless, the factors responsible for Nrg-1/Gdnf upregulation and their effects on glia cells are unknown. We investigated the effect on Nrg-1 and Gdnf expressions after stimulation of primary mouse microglia or astrocytes with various pro- and anti-inflammatory factors. Additionally, primary cells were incubated with NRG-1 and/or GDNF followed by determining the gene expression level of their receptors, chemokines, and other growth factors. We demonstrate that inflammatory stimuli have a distinct impact on the expression of Gdnf, Nrg-1, and their receptors in astrocytes and microglia. In microglia, LPS or simultaneous treatment with IFNγ plus TNFα led to downregulation of Nrg-1, whereas LPS treatment slightly increased Nrg-1 expression in astrocytes. Furthermore, Gdnf was slightly upregulated after TFG-ß treatment in microglia, while Gdnf was significantly upregulated after LPS treatment in astrocytes. In contrast, treatment with GDNF or/and NRG-1 did not alter any measured gene expression in microglia or astrocytes. Taken together, our in vitro studies show that Nrg-1, Gdnf, and their receptors are differently regulated in astrocytes and microglia upon inflammatory stimuli. The lack of response of astrocytes and microglia to NRG-1 and GDNF suggests that both factors exert their effects directly on neurons.

Fumaric Acids Directly Influence Gene Expression of Neuroprotective Factors in Rodent Microglia.

Dimethylfumarate (DMF) has been approved the for treatment of relapsing-remitting multiple sclerosis. The mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood, notably for brain resident cells. Therefore we investigated potential direct effects of DMF and MMF on microglia and indirect effects on oligodendrocytes. Primary rat microglia were differentiated into M1-like, M2-like and M0 phenotypes and treated in vitro with DMF or MMF. The gene expression of pro-inflammatory and anti-inflammatory factors such as growth factors (IGF-1), interleukins (IL-10, IL-1ß), chemokines (CCl3, CXCL-10) as well as cytokines (TGF-1ß, TNFα), iNOS, and the mannose receptor (MRC1) was examined by determining their transcription level with qPCR, and on the protein level by ELISA and FACS analysis. Furthermore, microglia function was determined by phagocytosis assays and indirect effects on oligodendroglial proliferation and differentiation. DMF treatment of M0 and M1-like polarized microglia demonstrated an upregulation of gene expression for IGF-1 and MRC1, but not on the protein level. While the phagocytic activity remained unchanged, DMF and MMF treated microglia supernatants led to an enhanced proliferation of oligodendrocyte precursor cells (OPC). These results suggest that DMF has anti-inflammatory effects on microglia which may result in enhanced proliferation of OPC.

Investigation of Cuprizone Inactivation by Temperature.

Animal models, such as cuprizone (bis-cyclohexanone oxaldihydrazone) feeding, are helpful to study experimental demyelination and remyelination in the context of diseases like multiple sclerosis. Cuprizone is a copper chelator, which when supplemented to the normal food of C57BL/6J mice in a concentration of 0.2% leads to oligodendroglial loss, subsequent microglia and astrocyte activation, resulting in demyelination. Termination of the cuprizone diet results in remyelination, promoted by newly formed mature oligodendrocytes. The exact mode of cuprizone's action is not well understood, and information about its inactivation and cleavage are still not available. The knowledge of these processes could lead to a better understanding of cuprizone's mode of action, as well as a safer handling of this toxin. We therefore performed experiments with the aim to inactivate cuprizone by thermal heating, since it was suggested in the past that cuprizone is heat sensitive. C57BL/6J mice were fed for 4 weeks with 0.2% cuprizone, either thermally pretreated (60, 80, 105, 121 °C) or not heated. In addition, primary rat oligodendrocytes, as a known selective toxic target of cuprizone, were incubated with 350 µM cuprizone solutions, which were either thermally pretreated or not. Our results demonstrate that none of the tested thermal pretreatment conditions could abrogate or restrict the toxic and demyelinating effects of cuprizone, neither in vitro nor in vivo. In conclusion, the current study rebuts the hypothesis of cuprizone as a heat-sensitive compound, as well as the assumption that heat exposure is a reason for an insufficient demyelination of cuprizone-containing pellets.

In vitro evaluation of physiologically relevant concentrations of teriflunomide on activation and proliferation of primary rodent microglia.

BACKGROUND: Teriflunomide, an inhibitor of dihydroorotate dehydrogenase, is thought to ameliorate multiple sclerosis by reducing activation-induced proliferation of lymphocytes, which is highly dependent on de novo pyrimidine synthesis. Nevertheless, its immunomodulatory effects on resident glial cells in the central nervous system are only poorly understood. METHODS: In this study, we employed physiologically relevant concentrations of teriflunomide and investigated its effects on survival, proliferation, activation, and function of primary rat microglia in vitro. RESULTS: We demonstrate that teriflunomide had no cytotoxic effect on microglia and had only a minor impact on microglial activation. In a concentration- and time-dependent manner, teriflunomide significantly downregulated surface expression of the co-stimulatory molecule CD86. Furthermore, in the highest concentration applied (5 µM), it slightly increased the expression of interleukin-10 in microglia in response to lipopolysaccharide. Treatment with low concentrations of teriflunomide (0.25-1 µM) did not have any impact on the activation or proliferation of microglia. At 5 µM concentration of teriflunomide, we observed a reduction of approximately 30 % in proliferation of microglia in mixed glial cell cultures. CONCLUSIONS: Taken together, our in vitro findings suggest that at higher concentrations, teriflunomide potentially exerts its effects by reducing microglial proliferation and not by modulating the M1-/M2-like cell differentiation of primary rat microglia. Thus, teriflunomide has no major impact on the plasticity of microglia; however, the anti-proliferative and minimal anti-inflammatory effects might be clinically relevant for immune modulation in the treatment of neuroinflammatory CNS diseases such as multiple sclerosis.